Poly-kringle plasminogen activator

ABSTRACT

Hybrid, third generation, plasminogen activators containing plural, heterologous polypeptide kringles prepared by recombinant DNA techniques as well as the genes coding for the activators, Vectors containing those genes and a method for using the plasminogen activators as thrombolytic agents, are disclosed.

This application is a continuation-in-part of Ser. No. 766,163, filedAug. 14, 1985, now abandoned, by P. P. Hung, N. K. Kalyan and S. L. Lee.

BACKGROUND OF THE INVENTION

Plasminogen activators are a class of serine proteases that convertplasminogen into plasmin. Plasmin degrades the fibrin matrix of bloodclots, thereby restoring the hemodynamic condition of an open vascularsystem after an internal vascular accident has produced thrombosis orthromboembolism. Vascular disease states involving partial or totalblockage of blood vessels which are amenable to treatment withplasminogen activators include stroke, pulmonary embolism, myocardialinfarction, as well as deep vein and peripheral artery obstructions.

There are two immunologically distinct types of plasminogen activatorsfound in human plasma and other body fluids--the urokinase-typeplasminogen activator (u-PA; M_(r), 55,000) and the tissue-typeplasminogen activator (t-PA; M_(r), 68,000). The activity of thetissue-type plasminogen activator is potentiated by fibrin. The enzymeacts at the site of a thrombus and demonstrates a higher affinity forfibri than does the urokinase-type plasminogen activator (Haylaeris etal., J. Biol. Chem., 257, 2912, 1982). Therefore, the tissue-typeplasminogen activator is considered to be the physiologically relevantthrombolytic agent.

Both activators, u-PA and t-PA, share the following common features: (1)they are synthesized as single chain proenzymes which can be cleaved byplasmin or trypsin, without disrupting their disulfide linked two-chainmolecular structure. Upon reduction, each plasminogen activator breaksdown into a heavy and a light chain (M_(r) 33,000 for u-PA; M_(r) 35,000for t-PA); (2) both enzymes are serine proteases which can beinactivated by serine-specific reagents such as diisopropylfluorophosphate; and (3) both enzymes contain a triple disulfide-linkedsequence of amino acids which form a loop or kringle in the molecule.Urokinase plasminogen activator has a single kringle. Tissue plasminogenactivator has two kringles connected by a hexapeptide linker sequence.These kringles are believed to be responsible for binding of the enzymesto fibrin (Thorsen, Biochem. Biophys. Acta., 393, 55, 1975).

The DNA sequence analysis and the amino acid sequence of t-PA isdisclosed by Pennica et al., Nature, 301, 214, (1983), Ny et al., Proc.Natl. Acad. Sci. U.S.A. 81 5355 (1984) and European Patent Application93,619 to Genentech Inc. The DNA sequence analysis and the amino acidsequence of u-PA is disclosed in European Patent Application 92,182 toGenentech Inc. and urokinase cDNA is discussed by Verde et al., Proc.Natl. Acad. Sci. U.S.A. 81 4727 (1984).

DESCRIPTION OF THE INVENTION

In accordance with this invention there is provided a group of hybrid,third generation, plasminogen activators containing plural, heterologouspolypeptide kringles. The polypeptides of this invention may containfrom 2 to 6 kringles. By heterologous kringles, applicants mean thepolypeptide product contains at least one kringle corresponding to thatfound in a different, naturally-occurring source or an additionalkringle structure common to and in addition to those found in a nativeplasminogen activator. It is understood that where a common kringlestructure is to be added to a native plasminogen activator to producethe hybrid plasminogen activators of this invention, the DNA ischemically synthesized and the DNA codon usage for production of thatcommon kringle must differ from that found in the DNA coding sequencefor the native plasminogen activator to avoid recombination (loopingout) while generating the desired amino acid sequence of the nativekringle. The kringles present in the hybrid plasminogen activators ofthis invention, although separately sharing areas of homology, areheterologous in nature and as a result thereof, the plasminogenactivators of this invention differ in their kringle combination fromany found in native plasminogen activators, either in amino acidsequence, number and/or size. In addition, this invention provides thegenes coding for the hybrid plasminogen activators and key fragmentsthereof, expression vectors for DNA production of the completepolypeptides as well as key fragments thereof, microorganisms or cellcultures transformed with the expression vectors and a method for usingthe hybrid plasminogen activators.

A kringle is triple looped polypeptide structure formed by threedisulfide bonds. Kringles vary in length from about 79 to 82 amino acidgroups. A high degree of sequence homology is shared among the singlekringle of human urokinase (Gunzler et al., Hoppe-Seyler's Z. Physiol.Chem. 363, 1155, 1982), the two kringles of human tissue plasminogenactivator (Pennica, et al. Nature, 301, 214, 1983), the two kringles ofhuman prothrombin (Walz et al., Proc. Nat'l. Acad. Sci. USA., 74, 1969,1977), and the five kringles of human plasminogen (Sottrup-Jensen etal., in Progress in Chemical Fibrinolysis and Thrombolysis (eds.Davidson et al.), 3, 191, 1978). The relative positions of the sixcysteins involved in the intra-kringle disulfide bridges are conservedin all kringles. The term, kringle(s) used in this application may betaken to mean any of such structure(s) in the above mentioned proteins.It is also understood that polymorphic forms in the kringle region ofthese proteins may exist in nature where one or more amino acids may beadded, deleted or substituted. Similar changes in a protein may also bebrought about in vitro with the advent of the present day technology ofpoint mutation of the gene or by chemical synthesis of the gene with anydesired sequence. These modified structure(s) are therefore alsoincluded in the term, kringle(s), used in this application.

The following description specifically illustrates the production of atriple kringle (tris-kringle) plasminogen activator, as well as atetra-kringle plasminogen activator. The methods employed arerepresentative of those applicable to the production of the otherpolypeptides of this invention.

The tris-kringle plasminogen activators of this invention, asconstructed by recombinant DNA techniques from appropriate geneticcoding sequences of urokinase and t-PA clones, offer the advantages ofincreased stability, increased binding affinity for fibrin and improvedhalf-life in vivo when compared to either of the native plasminogenactivators. These properties of the tris-kringle plasminogen activatorsprovide improved biological potency and improved shelf life. Thetris-kringle-PA molecule is easier to handle during production andpurification than native t-PA because the latter polypeptide, as foundin culture fluids and various purification stages, is accompanied by lowmolecular weight, heavy and light chain fragments. The tetra-kringleplasminogen activator as well as the other poly-kringle plasminogenactivators share these properties.

The tris-kringle plasminogen activator of this invention is constructedby combining the N-terminal portion of urokinase through its singlekringle region with that portion of t-PA beginning at or before thebeginning of the double kringle region via a suitable linker. The singlekringle of urokinase is known to precede the glycine residue at aminoacid position number 131. The double kringle of t-PA is known to beginat the cysteine following a threonine residue at position number 91.Thus, the N-terminal terminal portion of urokinase terminated at theglycine residue at number 131 is joined, optionally through a suitablelinker mimicking the hexapeptide link between the two kringles of t-PA,to a t-PA molecule from which the first 91 N-terminal amino acids havebeen deleted. The resulting hybrid molecule is larger than t-PA by 46amino acid residues providing a protein of about 73,000 M.W. (t-PA has amolecular weight of 68,000) depending upon the specific linker employed.Similarly, the single kringle of urokinase (UKKaa50-131), in conjunctionwith a kringle linker, is inserted into the t-PA polymer between aminoacids 91-92 or 261-262 to afford two different genes for production oftris-kringle plasminogen activators. Similarly, and by standardtechniques, the u-PA kringle may be inserted, with appropriate linkers,between the two t-PA kringles. In addition, the kringles found inprothrombin or plasminogen may be isolated and inserted in any of theposition of t-PA mentioned above. The construction of any of thepoly-kringle plasminogen activators of this invention follows the samebasic plan of isolating single or double kringle region from the proteinmentioned and inserting them into the backbone of the t-PA molecule.

The kringle linkers employed to join the single kringle portion ofurokinase (UKK) to the double kringle region of t-PA (t-PK1 and t-PK2)is a polypeptide containing 6 to 10 natural amino acid moieties.Preferably the kringle linker is selected to maintain a similar spatialarrangement to that which exists between the two kringles of t-PA. Assuch, the preferred linker is L-Ser-L-Glu-Gly-L-Asn-L-Ser-L-Asp becauseit is identical to that joining t-PK1 to t-PK2 in t-PA. However, thehexapeptide L-Thr-L-Asp-L-Ala-L-Glu-L-Thr-L-Glu represents anotherapplicable hexapeptide linker and any combination of L-Ala, Gly, L-Ser,L-Glu, L-Thr and L-Asp may be employed on the N-terminal or C-terminalends of the hexapeptide linker to provide a more open structure as ahepta-, octa-, nona- or decapeptide link between the t-PA kringles andthe UK kringle. Other linkers will be obvious to the chemist. Forsimplicity, the kringle linker specifically mentioned throughout therest of this application is limited to the preferred linker referred toabove.

The tris-kringle plasminogen activators are produced by limiteddigestion of urokinase and t-PA coding sequences with selectedrestriction enzymes to afford the desired u-PA and t-PA fragments. Thefragments are isolated by fractionation on agarose or acrylamide gels,ligated together and introduced into an appropriate vector or vectorsfor cloning and subsequent expression.

DESCRIPTION OF THE DRAWINGS

FIG. 1 presents schematic configurational drawings of three tris-kringleplasminogen activators and one tetra-kringle plasminogen activatorproduced by the methods disclosed in this application. The darkenedportion of the depicted structures 1 (a-c) defines that portion ofurokinase containing the urokinase kringle (UKK). The darkened portionof structure 1(d) defines the double kringle region of prothrombin (PTK1and PTK2). The abbreviatios tPK1 and tPK2 define the tissue plasminogenacivator kringle 1 and 2, respectively.

FIG. 2 depicts the restriction map of tissue plasminogen activatorrecombinant clone pWP-42.

FIG. 3 presents the technique followed in production of plasmid ptPBM-1from pWP-42. ptPBM-1 contains the genetic information needed to producethe complete t-PA molecule.

FIG. 4 presents the technique followed in production of plasmid pUKBMfrom plasmid pUK-53. pUKBM contains the genetic information needed toproduce the complete urokinase protein.

FIG. 5 presents a flow diagram of the method followed to produce thegene coding for the tris-kringle product depicted in FIG. 1(a).

FIG. 6 presents a flow diagram of the method followed to produce thegene coding for amino acids 51-131 of urokinase (u-PA⁵¹⁻¹³¹).

FIG. 7 presents a flow diagram of the method followed to produce thegene coding for the tris-kringle product depicted in FIG. 1(b).

FIG. 8 presents a flow diagram of the method followed to produce thegene coding for the tris-kringle product depicted in FIG. 1(c).

FIG. 9-9(g) presents the DNA sequence of the gene coding for the productof FIG. 1(a) with reference to the urokinase signal peptide region (20amino acids), the UKK region (amino acids 1-131 of urokinase), thehexapeptide linker and the remaining portion of the t-PA molecule (aminoacids 92-527).

FIG. 10-10(h) presents the DNA sequence of the gene coding for theproduct of FIG. 1(b) with reference to the t-PA signal peptide (35 aminoacids) the N-terminal portion of t-PA (amino acids 1-91), the UKK (aminoacids 50-131 of urokinase), the hexapeptide linker and the C-terminalportion of t-PA (amino acids 92-527).

FIG. 11-11(h) presents the DNA sequence of the gene coding for theproduct of FIG. 1(c) with reference to the t-PA signal peptide (35 aminoacids), the N-terminal portion of t-PA (amino acids 1-261), thehexapeptide linker, the UKK (amino acids 50-131 of urokinase) and theC-terminal portion of t-PA (amino acids 262-527).

FIG. 12 presents a flow diagram of the method followed to produce thegene coding for the double kringle region of prothrombin.

FIG. 13 presents a flow diagram of the method followed in producing thegene coding for the tetra-kringle product depicted in FIG. 1(d).

FIG. 14 presents a BPV-I based expression vector system designed forexpression of plasminogen activator Hybrid A (corresponding to FIG. 1(a)and Hybrid B (corresponding to FIG. 1(b)) genes in mammalian cell lineC-127 (mouse). The genes to be expressed, are inserted between the mousemetallothionein transcriptional promotor element and the SV40 earlyregion transcriptional processing signals.

FIG. 15 presents screening of PA-producing cells or foci on a fibrinagar plate by applying 10 μl of culture medium per well and incubatingat 37° C. till clear zones around the wells appear (lanes 2 to 7). Lane1 shows the standard t-PA with enzyme concentration (units/ml) from topto bottom: 500, 250, 100, 50, 25 and 0.

FIG. 16 presents a zymogram on a fibrin agar plate of Hybrid B, Hybrid Aand tissue type-plasminogen activators after separation of thehybrid-PA's by electrophoresis in a polyacrylamide gel (PAGE).

FIG. 17 presents an autoradiogram of ³⁵ S-labelled plasminogenactivators, Hybrid A and Hybrid B, after immunoprecipitation ofradio-pulsed medium with anti-t-PA antiserum and then electrophoresis onnon-reducing sodium dodecyl sulfate (SDS)/PAGE. Protein markers (rightlane) of known molecular weights were run concurrently.

METHODS AND MATERIALS (a) Enzymic Reactions

The restriction and DNA modifying enzymes were obtained from New EnglandBiolabs Inc., Beverly, MA or International Biotechnologies Inc., NewHeaven, CT. A typical restriction enzyme reaction was performed in atotal volume of 50 μl following the procedure(s) recommended by thesupplier of the enzyme.

A ligation reaction for the sticky end DNA is typically performed at 15°C. overnight in a buffered 20 μl solution containing 100-200 ng DNA and400 units of T4 DNA ligase (N.E. Biolabs.). For blunt end ligation, 4units of T4 RNA ligase (N.E. Biolabs.) are included in the abovereaction mixture. (Goodman, H. M., and MacDonald, R. J., Method.Enzymol. 68, 75, 1979). The buffer solution used is prepared as a stock10X solution; 0.5 m Tris®.HCl (pH 7.6), 0.1M MgCl₂ and 0.1M DTT(dithiothreitol).

(b) Synthesis of Oligonucleotides

All the oligonucleotides mentioned in this application were synthesizedby the phosphotriester method (Crea et al., Proc. Nat'l. Acad. Sci.(USA) 75, 5765, 1978) using the Gene Machine model 380A (AppliedBiosystems Inc., Foster city, CA). Before their use in ligationreactions, the oligomers were phosphorylated at the 5' end in a volumeof 50 μl containing 200-500 ng DNA, 10 units of T4 DNA kinase, 0.5 mMATP and kinase buffer (0.05M Tris.HCl, pH 7.6, 10 mM MgCl₂, 5 mM DTT)and incubated at 37° C. for 1/2 hour. For use as hybridization probes,oligomers were radiolabeled with 100 μCi gamma ³² P-ATP (5,000 C_(i)/mmol, Amersham, Arlington Heights, Il.) following the procedure ofMaxam, A. M. and Gilbert, W. Method Enzymol. 65, 499 (1980).

(c) Isolation of DNA Fragments

DNA fragments were first separated by electrophoresis through 0.5-1.5%agarose gel. Electrophoresis is carried out at about 100 volts for 2-4hours in Tris-Borate-EDTA (TBE) buffer (0.089M Tris, 0.089M boric acid,2 mM EDTA, pH 8.0). DNA bands are visualized under UV light by stainingthe gel in 0.5 μg/ml ethidium bromide solution (Sharp et al. Biochem.12, 3055, 1973). The agarose containing the DNA band is cut out with arazor. The DNA is electroeluted from the gel. (Maniatis et al. MolecularCloning, a Laboratory Manual, p. 164, 1982). The DNA is further purifiedby passing it through an Elutip-d® column (Schleicher and Schuell,Keene, NH). The DNA is precipitated with ethanol. After centrifugationin an Eppendorf microfuge for 15 minutes, the pellet is washed once with70% ethanol, dried under vacuum and dissolved in 50 μl deionized water.

(d) Miniplasmid DNA Preparation

About 2 ml of LB (Luria Bertani) medium containing appropriateantibiotics is inoculated with a single bacterial colony and isincubated at 37° C. overnight with vigorous shaking. About 1.5 ml of theculture medium is used to isolate plasmid DNA by the boiling methoddescribed in Maniatis et al., loc. cit. p. 366. The reset of the cultureis stored in 15% glycerol at -20° C. for later use. The DNA is dissolvedin 40 μl H₂ O containing 10 μg RNAse/ml. About 8 μl is sufficient forone restriction enzyme analysis.

(e) Large Scale Preparation of Plasmid DNA

Typically, one liter of LB medium is inoculated with a single bacterialcolony. After amplification of the plasmid DNA with chloramphenicol, thebacterial cells are harvested and lysed according to the boiling method(Holmes, D. S. and Quigley, M. Anal Biochem. 114, 193, 1981). Theplasmid DNA is further purified either by cesium chloride gradientcentrifugation or by column chromatography on a Sepharose 4B column(Pharmacia, Uppsala, Sweden) as described in Maniatis et al., loc. cit.pp. 93-96. A recovery of about 400 μg DNA per liter culture is routinelyobtained.

(f) Vectors

dG-tailed pBR322 plasmid DNA (Bethesda Research Laboratories, Inc.,Gaithersburg, MD) was used to clone the cDNA for t-PA and u-PA. Thedetailed molecular structure of pBR322 is described by Maniatis et al.,loc. cit. pp. 5 and 488. The E. coli strains used for transformationwith recombinatnt pBR322 were either HB101 or MM294 (Maniatis et al.,loc. cit. p. 504).

All of the subcloning of DNA fragments from t-PA and u-PA genes wereperformed in pUC plasmids--a series of pBR322 derived vectors containinglac Z and ampicillinase genes (Vieria, J. and Messing, J., Gene 19, 259,1982). In addition the plasmids also contain a sequence--multiplecloning or restriction site--in the lac Z, as shown below: ##STR1##Cloning in any of the 11 sites can be monitored by the appearance ofwhite recombinant colonies in the background of blue vector colonies onan indicator plate containing X-gal (5-bromo-4-Chloro-3-indolylβ-D-galactoside) (Ruther, Mol. Gen. Genetics 178, 475, 1980). The E.coli strain used for transformation with the recombinant pUC plasmid,was JM 103. The pUC plasmid and E. coli JM 103 were obtained fromPharmacia P-L Biochemicals, Milwaukee, WI.

(g) Host/vector System

1. Microbial System

The work described here was performed using the microorganisms E. coliK-12 strain JMM 103 (PL Biochemicals) and E. coli K-12 strain MM294(ATCC No. 33625). Other microorganisms which may be used in this processinclude other useful E. coli strains and Bacilli, such as Bacillussubtilis. All these microorganisms utilize plasmids that can replicateand express heterologous gene sequences.

The expression system in yeast employs a plasmid which is capable ofselection and replication in E. coli and/or yeast (Saccharomycescerevisiae). For selection in yeast, the plasmid contains the TRP 1 genewhich renders a transformed trp⁻ yeast strain (RH218) prototrophic fortryptophan. The yeast expression vector can shuttle in between yeast andE. coli. The plasmid has the following components: (1) a DNA segmentderived from PBR 322 containing the origin of replication and theampicillin resistance gene, (2) the yeast TRP 1 gene, (3) the yeast 2μDNA which enables the plasmid to replicate in yeast with high stability,(4) A promoter region from the yeast gene, such as alcoholdehydrogenase, α factor, glyceraldehyde-3-phosphatedehydrogenase, etc.,(5) translational start and transcriptional stop sequences which can beused for proper termination and polyadenylation of mRNA in theexpression system.

2. Mammalian Cell Culture System

Mammalian cell lines capable of the replication and expression of acompatible vector for the production of heterologous proteins can beused in the present invention. They are, for example: Cos-7, WI38, 3T3,CHO, Hela cells, and C127 cells. The vectors used contain (1) the originof replication derived from a virus (SV40, adeno, polyoma, BPV) orcellular chromosomal DNA, (2) a promoter, (3) the translationalinitiation signals, such as ribosomal binding sites, and (4) RNAprocessing signals, (RNA splicing, polyadenylation and transcriptionalterminator sequences). Specific examples of the expression vectorspresented here use a BPV viral replication origin, a mousemetallothionein promoter and SV40 RNA processing signals. The vector canalso be shuttled between mammalian cell culture and E. coli. It containsderivatives of PBR 322 sequences which provide selectable markers for E.coli ampicillin resistance as well as an E. coli origin of DNareplication. These sequences are derived from the plasmid pML-2d.

The edited hybrid plasminogen activator gene containing a Bam H1 stickyend is first inserted at the Bgl II site of plasmid 341-3 (Law MF etal., Md. Cell Biol. F 3, 2110, 1983) between the mouse metallothioneintranscriptional promotor element and the SV40 early regiontranscriptional processing signals. The complete BPV genome, obtainedafter digestion of plasmid 142-6 (ATCC No. 37134) with Bam Hl, isligated to the unique Bam Hl site. Plasmid 341-3 also contains pML2, apBR 322 derivative which allows plasmid replication in bacterial cells.The expression plasmid constructed herein can replicate in mouse C-127cells exclusively as an extrachromosomal episome. Transfected cells canbe selected for the transformed phenotype. Further modification of theexpression vector, such as by adding specific enhancer elements forhigher expression levels or inserting drug resistance (such as neomycinresistance) into the gene is also possible.

TRIS-KRINGLE PLASMINOGEN ACTIVATOR Tissue Plasminogen Activator (t-PA)

Messenger RNA

Total RNA was isolated by the isothiocyanate method (Maniatis et al.,loc. cit. p. 196) from normal human fibroblast cells (WI-38 cells),which had been stimulated by endothelial cell growth factor (ECGF) andheparin to produce t-PA. The same stimulated cells produce urokinase.Messenger RNA (mRNA) was obtained from the total RNA by chromatographyon an oligo-deoxythymidine (dT)-cellulose column (Aviv et al., Proc.Nat'l. Acad. Sci. USA, 69, 1408, 1972). Further fractionation of themRNA was performed by centrifugation in a 15-30% sucrose densitygradient and individual mRNA fractions were hybridized with ³² P-probes.(as described below). Fractions containing the t-PA message (ca. 20-24S)were pooled for use in the preparation of complementary DNA (cDNA).

Complementary DNA

The pooled mRNA (5 μg) described in the previous paragraph was used toproduce double stranded cDNA and the cDNA was homopolymer tailed withpolydeoxycytidylate (poly dC) using terminal nucleotide transferase. Theproduct was annealed with Pst 1 digested, polydeoxyguanylate (poly dg)tailed pBR322. The annealed DNA was used to transform competent E. coli294 cells which were cultured to produce about 10⁵ bacterial clones(Maniatis et al., loc cit., p. 229).

Screening and Identification of t-PA Clone

The following three oligonucleotides, after radiolabeling with ³² P-ATP,were used to screen the library of recombinant clones. These oligomerscorrespond to amino acid sequences, 34-39 (17 mer), 253-258 (18 mer) and523-527 (15 mer) of t-PA molecule (Pennica, D. et al., Nature, 301 214,1983) 17 mer: 5'-CCACTGTTGCACC-AGCA-3'; 18 mer:5'-CACATCACAGTACTCCCA-3'; 15 mer: 5'-CGGTCGCATGTTGTC-3'. About 20colonies exhibited moderate to strong homology with the pooled probes.Replating and rehybridization of these colonies gave 16 clones withpositive signals. Plasmid DNA prepared from these clones was blotted onnitrocellulose paper and hybridized with individual probes. Two clones(42 and 62a) hybridized to both the middle (18 mer) and 3' end (15 mer)probes. Enzymatic digestion of plasmid DNA with Pst 1 showed that cloneNo. 42 contained the biggest insert of greater than 2 kilobase (Kb) inthe form of three fragments of 1.1, 0.6 and 0.4 Kb. A completerestriction map of the clone (pWP 42) is depicted in FIG. 2 of thedrawings. This clone contains the full length sequence for the t-PAgene, containing 2600 bp, which includes the 5'- and 3'- untranslatedregions.

Editing of t-PA Gene

Approximately 10 μg of pWP 42 plasmid DNA was digested with 9 units XhoII at 37° C. for 2 hours. The reaction mixture was run on preparative1.2% agarose gel and a 1618 bp DNA fragment was isolated byelectrophoresis in agarose gel. After filling in cohesive ends with E.coli Polymerase 1 (Klenow fragment) and dNTPs (four deoxy nucleotidetriphosphate- dATP, dGTP, dCTP and dTTP) 1 μg of the so modified DNA wasligated overnight with 300 ng of phosphorylated Sal 1 linker. Afterphenol/chloroform extraction and ethanol precipitation, the DNA wasdigested with 50 U of Sal 1 for four hours and the reaction mixtureapplied to a preparative 1% agarose gel to isolate the desired DNAfragment.

The DNA with Sal 1 ends was ligated to Sal 1 cut pUC 13 and used totransform E. coli JM 103 cells and the cells were plated out onampicillin and X-gal plates. Eight ampicillin resistant, white colonieswere selected and grown to prepare a mini-plasmid preparation. Twoclones (ptPS34B and ptPS39) were found to contain the required DNAfragment. Ten μg of ptPS39 plasmid DNA digested to completion with BamHl and Nar 1 was run on preparative agarose gel to obtain a 1288 bpfragment coding for the C-terminal end of t-PA.

The 5' end of the t-PA gene was obtained by digestion of 10 μg of pWP 42with four units of Hga 1 at 37° C. for eight hours. A 515 bp fragmentwas isolated by electrophoresis in 1% agarose gel. The cohesive ends ofthis DNA fragment were filled in with DNA polymerase 1 (Klenow fragment)and dNTPs and the product was ligated to Sma 1 cut pUC 13. Aftertransforming E. coli JM 103 cells, approximately 75 ampicillinresistant, white colonies were obtained. Twenty four of these colonieswere grown to prepare a miniplasmid preparation. The miniplasmidpreparation was digested with Nar 1 and 17 clones were found to have therequired insert in either orientation. One clone (pt PHga 4) was grownin 1.0 liter of LB medium containing ampicillin to obtain a largequantity of plasmid DNA using the boiling method. The plasmid DNA, ptPHga 4, was digested with Bam Hl and Nar 1 and electrophoresed on 1.2%agarose gel to isolate a 434 bp DNA fragment coding for the N-terminalend of t-PA.

The 1288 bp DNA (300 ng) and 434 bp DNA (100 ng) were ligated overnightto obtain a 1722 bp DNA fragment. This DNA, after ligation wtih Bam Hlcut pUC 13 was used to transform E. coli JM 103 cells. More than 1000ampicillin resistant colonies were obtained. Plasmid DNA from twelvecolonies was prepared by the boiling method. The plasmid DNA wasidentified by cutting with each of Bam Hl, Nar 1 and Xho 11. All of theresulting plasmids were found to contain the desired 1,722 bp DNAfragment. One plasmid (pt PBM 1) was used for large scale plasmid DNApreparation. This plasmid, when cut with Bam Hl, gave rise to the 1,722bp DNA coding for the complete t-PA molecule. The pt PBM 1 clonerestriction map and a schematic diagram of its preparation is depictedin FIG. 3.

Urokinase Plasminogen Activator (u-PA) Screening and Identification ofu-PA Clone

The library of 10⁵ recombinant bacterial clones from which the t-PA genewas derived, supra, was screened with a radiolabelled 18 mer probe bythe method of Grunstein et al., Proc. Nat'l. Acad. Sci. USA, 72, 3961,(1975). The probe, synthesized by the standard phosphotriester methodusing a Gene Machine (Applied Biosystems), presents the oligomersequence-5'--GTA GAT GGC CGC AAA CCA--3'-corresponding to the middlepart of the urokinase gene (aa¹⁷³⁻¹⁷⁹). About 13 clones exhibited amoderate to strong hydridization signal. These clones were grown in 2 mlLB medium containing tetracycline and a miniplasmid preparation wasprepared. The miniplasmid preparation ws dissolved in 40 μL H₂ Ocontaining 10 μg/ml RNAse. About 8 μL of the DNA thereby produced wasdigested with one unit of Pst 1 and the product separated byelectrophoresis on 1% agarose gel. One clone (pUK 53) was found tocontain the largest insert of 1.7 Kb in the form of three inserts ofsizes 1.2, 0.4 and 0.1Kb long. The complete 3'- end nucleotide sequenceof urokinase was present in the Pst 1 cut 1.2 Kb DNA fragment. The 5'end sequence of the gene was discovered, through nucleotide sequencingby the Maxam and Gilbert method, Methods Enzymol., 65, 1499, (1980) tobe missing approximately 30 nucleotides corresponding to the first 10amino acids of the signal peptide coding region of the urokinaseprotein. Therefore, a duplex DNA sequence corresponding to the missingnucleotides was synthesized and ligated to the existing gene.

Editing of Urokinase Gene

The urokinase plasmid (pUK 53) DNA is cut with Nco 1 and Mst II and theproducts separated by electrophoresis on 1% agarose gel. A DNA fragmentof 1198 bp is isolated by electroelution. The 5' protruding end of theDNA fragment corresponding to the Nco 1 cut is made blunt ended byfilling in with dNTP's and E. coli DNA polymerase (Klenow fragment). TheDNA is then ligated to Sma 1 cut pUC 13 and the modified plasmid is usedto transform competent E. coli JM 103 cells. The Nco 1 site of theinsert is regenerated when the DNA is ligated to the Sma 1 site of pUC13. The cells are plated out on ampicillin and X-gal plates and aminiplasmid preparation is produced from white colonies. Digestion ofthe miniplasmid DNA preparation with Nco 1 and Sal 1 gives anapproximate 1200 bp DNA fragment. A large scale plasmid DNA preparationfrom a positive clone (pUKNM-3') is made and digested with Nco 1 and Sal1 to obtain a large amount of the approximate 1200 bp DNA fragment whichis separated by preparative agarose gel electrophoresis.

To provide the approximate 30 nucleotides corresponding to the first 10amino acids of the 5' signal peptide coding region of the urokinaseprotein, pUK 53 plasmid DNA is digested first with Pst 1 and a 400 bpDNA fragment was isolated. This DNA was then treated with ScrFl to yielda 242 bp fragment of DNA. The protruding ends of the DNA are filled inwith dNTP's and E. coli DNA polymerase 1 (Klenow fragment).

Two complementary oligonucleotide sequences, 38 and 42 bases in length,were synthesized on a Gene Machine to provide for missing amino aids (-9to -20) while keeping the proper translational reading frame andproviding a Sal 1 sequence on both ends of the DNA for subcloning in Sal1 cut pUC 13. The two oligomers are mixed in equimolar amounts in ligasebuffer (50 mM Tris.HCl, ph 7.6, 10 mM MgCl₂, 10 mM dithiothreitol) andheated to 80° C. for 5 minutes and allowed to cool to room temperaturefor about 1 hour. The thus formed duplex of the two complementarynucleotide sequences (about 1 μg) is ligated to about 300 ng of the 242bp DNA fragment in ligase buffer at 4° C. for 16 hours using 400 unitsof T4 DNA ligase. The ligated mixture is separated by electrophoresis on1.2% agarose gel and an approximate 320 bp DNA fragment is isolated byelectroelution. This fragment (about 20 ng) is ligated to 100 ng. of Sal1 cut pUC 13 and the vector is used to transform competent E. coli JM103 cells. The cells were plated out on ampicillin X-gal plates. Twelvewhite colonies were selected and grown to prepare a miniplasmidpreparation. The miniplasmid preparation is cut with Sal 1. One clone,containing the expected 320 bp DNA insert, is grown for large scalepreparation of plasmid DNA. The DNA is cut with Sal 1 and Nco 1 to yielda 260 bp DNA fragment upon preparative agarose gel electrophoresis.

The 260 bp DNA and 1200 bp DNA fragments, containing a common Nco 1restriction site at 333 bp position of the gene, are mixed in equimolaramounts for ligation. The ligated product is cut with Sal 1 and thereaction mixture separated by preparative 1% agarose gelelectrophoresis. A 1460 bp DNA fragment is isolated by electroelution.This DNA is ligated to Sal 1 cut pUC 13 and this plasmid is used totransform competent E. coli JM 103 cells which are plated out onampicillin and X-gal plates. Twelve white colonies were selected andgrown to prepare a miniplasmid preparation by the boiling method. Theminiplasmid preparation is cut with Sal 1 and one clone (pUKBM) wasfound to contain the desired 1460 bp DNA insert. pUKBM was grown inlarge volume to provide plasmid DNA. The oligonucleotide sequence fromthe 5' end containing the synthetic linker was sequenced by theMaxam-Gilbert method to confirm its authenticity.

The DNA insert in pUKBM plasmid was thereby established to contain thetranslational initiation codon AUG (met, -20 aa in the leader sequence)as well as the termination codon TGA. This complete gene codes for the20 amino acids of the signal peptide (-1 to -20) and the 411 amino acidsof mature urokinase protein.

EXAMPLE 1 (UKaa¹⁻¹³¹ -Ser-Glu-Gly-Asn-Ser-Asp)¹⁻⁹¹ t-PA

In compound (a.) shown in FIG. 1, the t-PA sequence containing thesignal peptide (a.a. -35 to -1) and the N-terminal peptide (a.a. 1 to91) regions are replaced by an amino acid sequence containing the signalpeptide (a.a. -20 to -1) and the first 131 amino acids of urokinase. Theu-PA sequence is joined to the first kringle (tPKl) of t-PA via aoligonucleotide sequence coding for the hexapeptideL-Ser-L-Glu-Gly-L-Asn-L-Ser-L-Asp.

About 10 μg of plasmid pUKBM is digested with Eco Rl and Mst 1 understandard conditions. The reaction mixture is electrophoresed through a1.2% agarose gel at 150 volts for 3 hours. After staining the gel withethidium bromide to visualize the DNA bands, a 452 bp DNA fragment isisolated and purified. This DNA fragment contains coding information forthe leader or signal peptide (20 amino acids) as well as N-terminal andkringle region (a.a. 1 to 131) of the urokinase gene.

In order to obtain the t-PA-des a.a 1-91 sequence, about 10 μg ofrecombinant plasmid ptPBM-1 is digested to completion with Ava II andelectrophoresed on a 1.2% agarose gel to isolate a 747 bp DNA fragment.The addition of an oligomer linker to this fragment followed bydigestion with Eco Rl to obtain a 354 bp DNA fragment is described inExample 2 and depicted in FIG. 7. The 354 bp DNA codes for thehexapeptide linker and the t-PA peptide (a.a. 92 to 204) - a 118 aminoacid sequence. Equimolar amounts of the 452 bp DNA fragment prepared inthe preceding paragraph and the 354 bp DNA fragment were ligated usingT4 DNA ligase at 15° C. for 16 hours. The reaction mixtue is separatedby electrophoresis on 1.2% agarose gel and a DNA band corresponding to asize of 806 bp was eluted out and purified. About 100 ng of 806 bp DNAfragment was ligated with about 300 ng of BAP (bacterial alkalinephosphatase) treated Eco Rl pUC 13 (Pharmacia P-L Biochemicals, Inc.Milwaukee, WI) in about 20 μl reaction volume. About 1/4 of the solutionwas used to transform competent E. coli JM 103 cells according to theprocedure of Viera, J. and Messing, J., Gene 19, 259, (1982).Miniplasmid DNA prepared from 12 recombinant white colonies, wasdigested with Eco Rl under standard conditions. One clone ptPUK-806,containing the required insert, was digested with Bam Hl and Nar 1 andseparated by electrophoresis on 1.2% agarose gel. A DNA bandcorresponding to a 519 bp fragment was cut, eluted and purified.Following the same procedure, a 1300 bp DNA fragment was obtained bydigestion of ptPBM-1 with Bam Hl and Nar 1. Equimolar amounts of 519 bpand 1300 bp DNA fragments were used for ligation following the standardprocedure of Goodman, H. M., and MacDonald, R. J., Method. Enzymol 68,75, (1979). The ligation mixture was extracted twice with aphenol:chloroform (1:1) mixture and the DNA was precipitated with twovolumes of absolute ethanol. After dissolving the pellet in 50 μl H₂ O,the DNA was digested wtih Bam Hl under standard assay conditions andseparated by electrophoresis on a 1% agarose gel. A DNA framentcontaining 1819 bps, was cut, eluted from the gel and purified. This DNAfragment contains all the coding information required for the signalpeptide (20 amino acids) and the mature hybrid or tris-kringle PAmolecule of 573 amino acids which corresponds to (UKaa¹⁻¹³¹-Ser-Glu-Gly-Asn-Ser-Asp)¹⁻⁹¹ -t-PA depicted in FIG. 1(a).

The tris-kringle gene is then ligated to Bgl II cut expression vectorsbovine papalloma virus (BPV) which serves as the complete expressionvector. Conventional culture yields the tris-kringle plasminogenactivator of FIG. 1(a).

Construction of Urokinase Kringle Sequence

In Examples 2 and 3, only the kringle part (a.a. 50-131) of urokinase isutilized and is inserted either before or after the double kringleregion of t-PA. FIG. 6 depicts the construction of a nucleotidesequence, coding for a.a. 51-131 from the recombinant plasmid pUK 53.There is a convenient restriction site, Mst 1, just after the nucleotidesequence corresponding to amino acid 131. However, none could be foundaround a.a. 50. Thus, a scheme for the creation of a restriction sitearound a.a. 50 (Nde 1 in this example) was formulated as shown in FIG.6. The kringle region of urokinase corresponds to the nucleotidesequence from bp 284 (a.a. 50) to bp 530 (a.a. 131).

About 10 μg of pUK 53 plasmid DNA was digested to completion with Sca 1which cuts at bp 204 in the urokinase sequence. After phenol extractionand ethanol precipitation, the DNA pellet was dissolved in 50 μl ofbuffer solution (10 mM CaCl₂, 12 mM MgCl₂, 0.2M NaCl 1, 20 mM Tris.HCl(pH 8.0), 1 mM EDTA). To the reaction mixture was added 1 μl (2 units)of nuclease Bal 31 and the mixture was incubated at 30° C. for 15seconds (Legerski, R. J., J. L. Hodnett, and H. B. Gray, Jr., NucleicAcid Res. 5, 145, 1978). The reaction was stopped by the addition of 5μl of 0.4M EGTA. This reaction time was found to be sufficient to removeabout 80 bp from each end of the DNA fragment. After phenol extractionand ethanol precipitation, the DNA was ligated to an oligonucleotidelinker (10 bp) under standard reaction conditions. The oligomer linker(Eco Rl/Nde 1 linker) with the sequence, TGGAATTCCA, was designed tocreate an Nde 1 site (CATATG) when ligated to the DNA fragment endcontaining the sequence, TATG (corresponding to a.a. 51). In addition,the restriction site, Eco Rl, was built into the linker to provide forsubsequent cloning in a pUC 13 vector. After phenol extraction andethanol precipitation, the DNA was digested wtih Eco Rl and separated byelectrophoresis on 1% preparative agarose gel. A DNA band correspondingto 340 bp, was cut, eluted and ethanol precipitated. About 40 ng of thisDNA was ligated with about 0.4 μg of Eco Rl cut pUC 13 vector DNA andused to transform competent E. coli JM 103 cells (Maniatis et al., loc.cit. p. 250). About 1,000 recombinant colonies were obtained from 10plates. The bacterial colonies were replica-plated on nitro-cellulosepaper, and screened by in situ hybridization using a radioactiveoligonucleotide probe (Grunstein et al. Proc. Nat'l. Acad. Sci USA 72,3961, 1975). The oligonucleotide probe used was 18 bp long(TTCCATATGAGGGGAATG) and contains the first five nucleotides from theEco Rl/Nde 1 linker and the next 13 bases from the urokinase sequencecorresponding to a.a 51 to 54. About 12 clones showed a moderate tostrong signal on X-ray film. Miniplasmid DNA prepared from these 12clones was digested with Nde 1 and separated by electrophoresis on 1%agarose gel. One clone, pUKKNd 16, was found to contain the newlygenerated Nde 1 site. This plasmid DNA, after digestion wtih Nde 1 andMst 1, was separated by electrophoresis on 1.4% agarose gel to obtain a246 bp DNA fragment. This DNA fragment contains the urokinase nucleotidesequence coding for a.a. 51 to 131. The DNA sequence for the missinga.a. 50 (Cys) is incorporated into the oligomer linker as shown in FIG.7 and 8.

EXAMPLE 2 91-(UKaa⁵⁰⁻¹³¹ -Ser-Glu-Gly-Asn-Ser-Asp)-92-t-PA

The urokinase kringle sequence as shown in FIG. 1(b) was inserted beforethe double kringle region of t-PA i.e. between a.a. 91 and 92. Asdescribed in the preceding paragraph, Nde 1 and Mst 1 digestion ofpUKKNd 16 plasmid DNA gave rise to a 246 bp sequence which correspondsto a.a. 51 to 131 of urokinase. The two ends of the UK kringle sequence(246 bp) were inserted into the t-PA gene between nucleotide no. 462(a.a. 91) and 463 (a.a. 92) through the use of two oligonucleotidelinkers. The procedure followed is shown in FIG. 7.

About 50 μg of ptPBM-1 plasmid DNA was digested with Eco Rl and a 740 bpDNA fragment was isolated by electrophoresis in 1% agarose gel. This DNAwas then digested partially with Sau 961 (isoschizomer of Asu 1) for theisolation and purification of a 385 bp DNA fragment.

Two complementary oligonucleotide sequences were synthesized by thephosphotriester method (Crea et al., Proc. Nat'l. Acad. Sci (USA) 75,5765, 1978) which code for amino acid 91 (Thr) of t-PA and amino acid 50(Cys) of the urokinase kringle. ##STR2## The oligonucleotide linker isflanked by Asu 1 and Nde 1 restriction sites. Only the upperoligonucleotide (GGCCACCTGC) was phosphorylate at its 5' end.

About 1 μg of the 385 bp DNA fragment was ligated overnight withapproximately 1 μg of oligomer linker. After phenol extraction andethanol precipitation, the DNA was digested for 2 hours with Eco Rl andseparated by electrophoresis on 1.2% agarose gel to obtain a 394 bp DNAfragment. This DNA fragment contains the nucleotide sequences coding forthe signal peptide (35 a.a.) and N-terminal peptide a.a. 1-91 of t-PA.In addition, it also restores the DNA sequence for a.a. 50 (Cys) of theUK kringle not present in its 246 bp DNA fragment (FIG. 6).

In order to obtain the C-terminal t-PA sequence, amino acids 92 onward,about 50 μg of ptPBM-1 plasmid DNA was digested with Ava II and a 747 bpDNA fragment was isolated from 1% agarose gel. Two complementary DNAfragments of 27 and 30 bp long were synthesized by the phosphotriestermethod. As shown in FIG. 7, this DNA linker codes for the hexapeptideSer-Glu-gly-Asn-Ser-Asp and also restores the missing a.a. 92 to 94(Cys-Tyr-Glu) in the Ava II cut 747 bp DNA. Only the lower oligomer (30mer) is phosphorylated at its 5' end. About 1 μg of 747 bp DNA and 1 μgof oligomer linker were ligated overnight at 15° C. After phenolextraction and ethanol precipitation, the DNa was digested for 2 hourswith Eco Rl to obtain a 354 bp DNA fragment from 1.4% agarose gel. About500 ng of each of the three DNA fragments, 394 bp, 246 bp (UK Kringle)and 354 bp, were ligated overnight using T4 DNa ligase. After phenolextraction and ethanol precipitation, the DNA was digested with Eco Rland a 994 bp DNa fragment was isolated and purified from the 1% agarosegel. This DNA, after ligation with an equimolar amount of Eco Rl cut pUC13 vector, was used to transform competent E. coli JM 103 cells.Miniplasmid DNA preparations from 12 recombinant clones were digestedwith Eco Rl. One clone containing the required insert of 994 bp, wasgrown in 1 liter LB medium for large scale preparation of plasmid DNA.About 10 μg of this plasmid DNA was digested with Bam Hl and Nar 1 and aDNA fragment corresponding to 700 bp size was purified from the 1%agarose gel.

The 3' end of the t-PA gene was obtained by digestion of about 10 μg ofptPBM-1 plasmid DNA with Bam Hl and Nar 1. After separating the reactionmixture by electrophoresis on 1% agarose gel, an approximate 1300 bp DNAfragment was isolated by electroelution and purified.

Approximately equimolar amounts of the two DNA fragments of 700 bp and1300 bp size, were ligated overnight and a 2000 bp DNA fragment wasrecovered from 1% agarose gel. This DNA, flanked by the restrictionenzyme Bam Hl sequence, is inserted at the Bgl II site of the BPVexpression vector. This DNA codes for a protein containing a total of650 amino acids-35 amino acids for the signal peptide and 615 aminoacids for the mature protein. Conventional culture, recovery, isolationand purification techniques yield the tris-kringle plasminogen activatorof FIG. 1(b).

EXAMPLE 3 261-(Ser-Glu-Gly-Asn-Ser-Asp-UKaa⁵⁰⁻¹³¹)-262-t-PA

In this example, the urokinase kringle sequence is inserted just afterthe double kringle region of the t-PA gene i.e. between the sequencescorresponding to a.a. 261 (Cys.) and 262 (Ser). FIG. 8 shows the schemedetailing the various steps involved in the production of thistris-kringle PA.

About 10 μg of pWP 42 plasmid DNA was digested with Hga 1 and a 400 bpDNA fragment was isolated from 1% agarose gel. This DNA fragmentcontains the sequence corresponding to part of the kringle region oft-PA, i.e., amino acids 135-261. It should be noted that the doublekringle region of t-PA ranges from amino acids 92 to 261, with ahexapeptide (amino acids 174-179) joining the two kringles. Anoligonucleotide linker consisting of two DNa sequences-24 mer and 21 meras depicted in FIG. 8-was synthesized using the phosphotriester method.This oligomer linker codes for the hexapeptide linker as well as formissing amino acid (Cys) of the UK kringle and is flanked by restrictionenzyme sequences for Hga 1 and Nde 1. Only the upper oligonucleotide, 23mer is phosphorylated at its 5' end and it is ligated to the Hga 1 endof the 400 bp DNA fragment from t-PA. The 421 bp DNA product is isolatedfrom preparative 1% agarose gel.

The post kringle part of t-PA was obtained by digestion of 10 μg of pWP42 plasmid DNA with Rsa 1 followed by isolation of 501 bp DNA from 1.2%agarose gel. This DNA represents a.a. 269 to 435 of the t-PA molecule.Two complementary oligonuclelotide sequences of 22 bases as depicted inFIG. 8 were synthesized by the phosphotriester method. This DNA linker,when ligated to the 501 bp DNA at its 5' end restores the missing aminoacids from 262 to 268.

Approximately 1 μg of the 501 bp DNA and 1 μg of the linker DNA wereligated overnight and a DNA band corresponding to 523 bp size waspurified from 1% agarose gel.

The three DNA fragments of sizes 421 bp, 246 bp (UK Kringle) and 523 bp,were ligated in approximately equimolar amounts at 15° C. for 16 hours.After phenol extraction and ethanol precipitation, the DNA was cut withEco Rl and a DNA fragment of 741 bp was isolated. This DNA, flanked bytwo Eco Rl restriction sites, was amplified in pUC 13 vector system asdescribed above.

10 μg of ptPBM-1 plasmid DNA in which the Ego Rl site in multiplecloning site had been removed, was digested to completion with Eco Rland the larger vector DNA fragment of about 4.0 Kb was isolated form 1%agarose gel. About equimolar amounts of Eco Rl cut vector DNA and the741 bp DNA was ligtaed and the product used to transform competent E.coli JM 103 cells. Miniplasmid DNA, prepared from 12 recombinant clones,was digested with Bam Hl to look for the desired insert of about 1.8 Kb.The correct orientation of the 741 bp DNA in the insert was determinedby digestion of the plasmid DNA with Nar 1 and Mst 1. One clone,PtPUHYC, when digested with Nar 1 and Mst 1 was found to contain afragment of approximately 700 bp in correct orientation.

The 1.8 Kb DNA, obtained by digestion of ptPUHYC plasmid DNA with BamHl, contains the nucleotide sequence coding for a protein of 650 aminoacids-35 amino acids for the signal peptide and 615 amino acids formature protein corresponding to the product of FIG. 1(c).

The tris-kringle gene is then ligated to Bgl II cut bovine papallomavirus (BPV) which serves as the complete expression vector. Conventionalculture yields the tris-kringle plasminogen activator of FIG. 1(c).

TETRA-KRINGLE PLASMINOGEN ACTIVATOR Prothrombin cDNA:

A cDNA clone for the prothrombin gene was isolated from the human livercDNA library following the procedure of Friezner et al., Biochemistry,22, 2087, 1983. The clone, pPTR, contains complete coding informationfor the mature protein of 579 amino acids. The double kringle region ofprothrombin extends from amino acid 65 (bp 319) to amino acid 248 (bp840), a 184 amino acid peptide. Each of the two kringles, PTK1 (aminoacids 65-143) and PTK 2 (amino acids 170-248) are 79 amino acids longand are joined by a peptide of 26 amino acid length (amino acids144-169).

Preparation of Prothrombin Double Kringle Sequence

The DNA sequence representing the double kringle region was isolatedfrom the prothrombin cDNA in two steps (FIG. 12).

In the first step, 10 μg of pPTR plasmid DNA was digested with Ava 1 anda 706 bp DNA was isolated from 1% agarose gel. This DNA was treated withBal 31 for 7.5 seconds to remove about 38 bp from either end of the DNA(Legerski et al., Nucleic Acid Res., 5, 145, 1978). After phenolextraction and ethanol precipitation, the DNA was ligated to an Eco Rllinker, GGAATTCC, at 4° C. for 15 hours. This linker, when ligated toDNA ending with sequences CTGAG or TGAG (amino acid 67, Glu) willproduce a new restriction sequence for Mst II (CCTGAGG) at theN-terminal of the double kringle (amion acid 67). After cutting with EcoRl, a DNA fragment of 630 bp size was isolated. This DNA, after ligationin equimolar amounts with Eco Rl/pUC 13, was then used to transformcompetent E. coli JM 103 cells. Initial screening for the desired clonewas performed by in situ hybridization with a ³² P labeled probe,GAATTCCTGAGGGTCTG, containing nucleotide sequences for amino acids 66 to68. Twelve clones, exhibiting a strong signal on X-ray film, were grownfor a miniplasmid preparation. After digestion of plasmid DNA with MstII and Bam Hl, one clone, pPTK- b 5' was found to contain the requiredinsert of about 630 bp and also the newly created Mst II site at 322 bp.

The second step involved the editing of the 3' end of the kringleregion, around amino acid 248, using a similar approach as describedabove. About 10 μg of pPTK-5' plasmid DNA was digested to completionwith Bam Hl. The DNA was then treated with Bal 31 for 15 seconds at 30°C. to remove about 82 bp from both ends of the DNA. After phenolextraction and ethanol precipitation, the DNA was ligated to a EcoRl/Fsp 1 linker, CGCAGAATTCTGCG. As the name suggests, the linkergenerates an Fsp I sequence (TGCGCA) at any DNA sequence ending in TG-.After cutting thoroughly with Eco Rl, a 546 bp DNA was isolated from1.2% agarose gel and then ligated to Eco Rl cut pUC 13. The recombinantplasmid was then used to transform competent E. coli JM 103 cells. About1000 recombinant clones were screened in situ using a ³² P-labelledoligomer with the sequence CTCAACTATTGCGCAGAA (amino acids 245 to 248 ).Plasmid DNA prepared from 12 potential clones, was cut with Mst II andFsp 1 and run on a 1% agarose gel. One clone, pPT2K, was found tocontain the required insert of 546 bp size. This DNA codes for a totalof 182 amino acids, amino acids 67-248, of the double kringle region.The nucleotide sequence for the remaining two amino acids, at positions65 (Cys) and 66 (Ala), are added via the oligomer linker as shown inFIG. 13.

EXAMPLE 4 91(PTKaa⁶⁵⁻²⁴⁸ -Ser-Glu-Gly-Asn-Ser-Asp)-92-t-PA

The double kringle sequence of prothrombin, amino acids 65-248, wasinserted before the double kringle region of t-PA (i.e. between aminoacids 91 and 92) to give rise to tetra kringle-PA. As described in thepreceding paragraph, Mst II+FspI or Eco Rl digestion of pPT2K plasmidDNA gives rise to a 546 bp DNA fragment which codes for amino acids 67to 248 of prothrombin. Through the use of two oligonucleotide linkers,the two ends of the 546 bp DNA were inserted into the t-PA gene betweennucleotide 462 (amino acid 91) and 463 (amino acid 92). The schemefollowed is shown in FIG. 13.

The 354 bp DNA, containing the nucleotide sequence for the peptidelinker-Ser-Glu-Gly-Asn-Ser-Asp as well as for the amino acid 92 to 204of t-PA, was prepared as described in Example 2 and FIG. 7. Equimolaramounts of the 354 bp DNA and 546 bp DNA (prothrombin kringles) wasligated at 4° C. for 16 hours. The DNA was cut with Eco Rl and a 900 bpDNA fragment was isolated from agarose gel. This DNA after ligation toEco Rl cut pUK 13 vector was used to transform E. coli JM 103 cells.Miniplasmid preparation obtained from 12 recombinant clones weredigested with Eco Rl and Mst II. One clone, pPT2KTPK, was found tocontain the required insert of 900 bp. This DNA codes for a total of 300amino acids, 182 amino acids for prothrombin (amino acids 67-248), ahexapeptide sequence and 112 amino acids for t-PA (amino acids 92-204).

The preparation of 385 bp DNA obtained by digestion of ptPBM-1 plasmidwith Eco Rl and Asu 1 is described in FIG. 7. Two complementaryoligonucleotide sequences of 12 bp each, were synthesized by thephosphotriester method. This linker restores the missing amino acids,Cys and Ala, of the prothrombin kringle region and is flanked by the Asu1 and Mst II recognition sequences. About 500 ng of DNA (385 bp) wasligated with 1 μg of phosphorylated linker as shown in FIG. 13. Aftercutting with Eco Rl, a 402 bp DNA fragment was isolated. About equimolaramounts of 402 bp DNA and 900 bp DNA were ligated, cut with Eco Rl and a1302 bp DNA fragment was isolated from the 1% agarose gel. This DNA wassubcloned in Eco Rl cut pUC 13 vector. Plasmid DNA, isolated from 12recombinant clones, was digested with Eco Rl. One clone, pNPT2KPAK,containing the required insert, was grown for large scale preparation ofplasmid DNA. The plasmid DNA was digested to completion with Bam Hl andthen partially with Nar 1 to obtain a 986 bp DNA fragment. Similarly, a1300 bp DNA fragment was obtained from digestion of ptPBM-1 with Bam Hland Nar 1. the two DNA fragments, 986 bp and 1300 bp, in equimolaramounts were ligated, cut with Bam Hl and a 2286 bp DNA fragment wasisolated from 1% agarose gel electrophoresis. The DNA, flanked by theBam Hl sequences, was inserted at the Bgl II site of the BPV expressionvector. This DNA codes for a total of 752 amino acids-35 amino acids forthe signal peptide and 717 amino acids for the mature protein. Themature protein, with an estimated molecular weight of about 92,000contains 184 amino acids from the prothrombin double kringle region, ahexapeptide linker and complete t-PA sequence of 527 amino acids.

Expression and Biochemical Characterization of Hybrid PlasminogenActivators

Two of the hybrid plasminogen activators (h-PA) shown in FIG. 1(a) andFIG. 1(b) were prepared for expression in the BPV-I based expressionvector system. These h-PA's are throughout this application referred toas Hybrid A and Hybrid B, respectively.

The complete gene sequences, flanked by BamHI for Hybrid A (1.8 Kb, FIG.5) and Hybrid B (2.0 Kb, FIG. 7) were inserted into the Bgl II cutplasmid p341-3 (for details see Methods and Materials). Miniplasmidpreparations from 12 recombinant clones were prepared and digestedindividually with Nar I, Bgl II or BamHI and Ava I. This was used toconfirm the presence of hybrid genes as well as to determine theorientation of the insert. Only one orientation of the gene, i.e., inthe direction of the metallothionein promotor, is desirable, because itplaces the expression of the gene under the control of that promotor. Inaddition, the SV40 poly-A sequence located just behind the gene, wouldprocess the RNA transcript of the gene by polyadenylation at its 3'-endfor efficient translation of the gene. Two recombinant clones pHybAMT-43 (for Hybrid A) and pHyb BMT-50 (for Hybrid B) were obtained.

About 1 μg of the plasmid DNA obtained from the above mentioned clones,was digested with BamHI and then dephosphorylated with bacterialalkaline phosphatase. To this was inserted a complete 8.0 Kb BamHI cutBPV-I genome. Two expression plasmids pHyb-AMTBPV-438 andpHyb-BMTBPV-504 (simply referred to as p438 and p504) containing thegenes coding for Hybrid A and Hybrid B, respectively, were obtained. Acomplete map showing the relative positions of various components of theexpression plasmids is shown in FIG. 14.

The two expression plasmids containing the genes encoding for hybridt-PA/urokinase molecules were transfected into mouse C127 cells by thecalcium phosphate precipitation method (Graham et al., Virology, 52, 456(1973)). Foci of morphologically transformed cells were subsultured andscreened for gene expression. Initial screening for fibrinolyticactivity in the medium was done on a fibrin-agar plate as shown in FIG.15(a) (Ploug et al. Biochim. biophys. Acta, 24, 278 (1957)). From eachtransfection, an average of 50% of foci transformed with p504 (Hybrid B)and 5% of foci transformed with p438 (Hybrid A) showed positivefibrinolytic activity in the culture medium. Several high producers wereselected and cell lines were expanded from individual foci. The two celllines with which most of the preliminary biochemical and immunologicalcharacterizations of the gene products were performed were labeled clone5A5 for Hybrid B and clone 16Cl for Hybrid A.

Biochemical Characterization

The enzymatic activities of the hybrid molecules were assessed byfibrinagar assay and amidolytic activity assay using syntheticsubstrates S-2444 and S-2251 (Shimoda et al., Thromb. Haemostas., 46,507 (1981)). Aproximately 1-5 units/μl active enzyme were secreted intothe medium in 16 and 18 hours. Natural t-PA and urokinase aresynthesized as precursors and secreted from the cells after the signalsequences are cleaved off to become the mature protein. Furthermore,both t-PA and urokinase are glycosylated. To determine whether theh-PA's secreted by the transfected mouse cells were processed in asimilar fashion as natural t-PA and urokinase, the hybrid molecules wereanalyzed by SDS/polyacrylamide gel (PAGE) electrophoresis followed byfibrin-agar overlay (Granelli-Piperno et al., J. Exp. Med., 148, 223,(1978)). As shown in FIG. 15(b), the active enzyme from HybridB-containing medium has a molecular weight of 76,000 and Hybrid A,71,000. These are in good agreement with the molecular weight calculatedfrom the inserted gene.

Hybrid B was purified from harvest medium in a manner similar to thatused for t-PA purification. Amino acid sequence analysis indicated thatthe N-terminal of Hybrid B is correctly processed, having the sequenceidentical to the N-terminal region of mature t-PA: Ser-Tyr-Gln-; inaddition, an N-terminal sequence, Ile-Lys-Gly-, corresponding to theamino acid at the activation cleavage site Arg-Ile was present. It isconcluded that Hybrid B purified from the harvest medium existed mainlyas an activated, two chain form. By addition of a protease inhibitor(Aprotinin) to the harvest medium, a single chain h-PA molecule isobtained.

Immunoprecipitation followed by SDS/PAGE of ³⁵ S-labeled harvest mediashowed that, under non-reducing conditions, bands at the positioncorresponding to an apparent molecular weight of 71,000-76,000 wereobserved in the sample from cell line 5A5 (Hybrid B) and 16Cl (HybridA), respectively, but not in the control sample. The fibrinolyticactivity of the culture media from 5A5 cells as well as purified t-PA(American Diagnostics, Inc., Greenwich, CT) was neutralized by anti-t-PAantiserum, but not by anti-urokinase antiserum, suggesting that althoughthe Hybrid B contains a urokinase kringle in addition to t-PA, theprotease domain of Hybrid B was recognized and neutralized by anti-t-PA.Anti-urokinase antibody may bind to the urokinase kringle portion of thehybrid molecule, but this binding, if any, does not interfere with theproteolytic activity conferred by the protease domain at the C-terminusof the hybrid molecule.

The poly-kringle plasminogen activators of this invention are used intreatment of vascular accidents in mammals in the same manner andthrough the same delivery vehicles as t-PA itself. Thus the poly-kringleplasminogen activators of this invention may be formulated intopharmaceutical compositions by dissolving or suspending the polypeptidesin suitable pharmaceutically acceptable vehicles known to the art asapplied to t-PA. Administration to a mammal in need thereof byintravascular injection or infusion is conducted following techniquesalready established with t-PA itself. An intravenous primary dose ofabout 440 IU/kg body weight is normal, followed by continuing infusionof about 440 IU/kg/hr for about 6 to 12 hours is conventional practicewhen using t-PA.

What is claimed is:
 1. A human tissue plasminogen activator hybridcomprising at least both kringle regions of human tissue plasminogenactivator and one or two heterologous kringles selected from the groupconsisting of the human urokinase kringle and either of the humanprothrombin kringles, wherein said hybrid possesses the fibrinolytic andamidolytic activities of native human tissue plasminogen activator. 2.The plasminogen activator of claim 1 which is 91-(PTKaa⁶⁵⁻²⁴⁸-Ser-Glu-Gly-Asn-Ser-Asp)-92 t-PA.
 3. The plasminogen activator of claim1 which is (UKaa¹⁻¹³¹ -Ser-Glu-Gly-Asn-Ser-Asp)¹⁻⁹¹ t-PA.
 4. Theplasminogen activator of claim 1 which is 91-(UKaa⁵⁰⁻¹³¹-Ser-Glu-Gly-Asn-Ser-Asp)-92 t-PA.
 5. The plasminogen activator of claim1 which is 261-(Ser-Glu-Gly-Asn-Ser-Asp-UKaa⁵⁰⁻¹³¹)-262 t-PA.